The first response to infection, which includes Toll-like receptor (TLR) mediated processes, is crucial for fighting pathogen invasion and for potentiating the adaptive immune response. The zebrafish is an ideal vertebrate model to study innate immune response to infection through the TLR pathways. As a lower vertebrate, the zebrafish has a simpler immune system than mammals, and in the first 30 - 60 days of development the zebrafish relies solely on the innate immune response for protection against pathogens. This provides a window in time in which it is possible to characterize innate immunity in an organism that is eating, swimming, and otherwise interacting with its environment, and can easily be exposed to pathogens to initiate an immune response. Our laboratory has conducted extensive analysis of TLR-mediated signaling in the zebrafish. In addition, bacterial and viral infection models have been developed in our lab. During the course of our studies, we discovered that the zebrafish TICAM1 orthologue, a TLR adaptor protein in mammals, can activate zebrafish type I interferon as it does in mammals, but through an alternative mechanism. Thus, these orthologues demonstrate functional, but not mechanistic conservation. Significantly, this implies that novel TLR-mediated signaling pathways may be active in innate immunity. We hypothesize that zebrafish TLR signaling may be mechanistically divergent from mammals. Furthermore, these differences may lead to the discovery of novel TLR pathways in higher vertebrates, such as humans. The elucidation of these novel pathways may provide insight into alternative therapies and treatments for infectious disease. In this proposal, we intend to fulfill the following SPECIFIC AIMS: 1. Analyze the role of zebrafish adaptor proteins in immune defense against pathogens 2. Determine the critical domains of zebrafish adaptor proteins and characterize the TLR adaptor protein deletion mutants through in vivo functional analysis 3. Identify novel TLR signaling pathway components that generate an immune response in vivo. Infectious diseases continue to play a major role in the human condition worldwide and as a result vaccines, antibiotics, and intervention strategies are continually being developed. These methods have largely proven effective; however, due to microbial adaptation and environment- triggered emergence of new diseases, it is critical to investigate alternative methods for controlling infectious diseases. A critical focus of this investigation involves the immune system itself, specifically ways in which its function can be augmented, extended, and prolonged. Recent recognition of the intrinsic importance of the innate immune response beyond its regulatory role in the subsequent adaptive immune response suggests that this system plays a crucial role in protection against infectious diseases. [unreadable] [unreadable] [unreadable]